Human Cdc7-related Kinase Complex

Abstract

huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D., Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.

Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

“…Annotation of the human Mcm2 amino acid sequence has been recently updated in its N terminus, and here we refer to its new Swiss Prot accession number, P49736. We and others have previously shown that an Mcm2 N-terminal fragment, corresponding to amino acids 10 -294, is a good substrate for both Cdc7 and CDKs (9,18,20). We extended these observations and also found that CK2 kinase, which has been proposed to modulate DNA replication by phosphorylating multiple substrates (17), efficiently uses the Mcm2 N-terminal fragment as a substrate.…”

“…Previous work has suggested that Cdk2 phosphorylation of Mcm2 stimulates phosphorylation by Cdc7 (9). Importantly, Cdc7-dependent phosphorylation was primarily measured as an increased amount of Mcm2 with altered mobility in SDS-PAGE.…”

“…Reversing the order of the kinases appeared to be detrimental to DNA replication in this system (12). Finally, in vitro phosphorylation assays using purified human recombinant proteins have indicated that pre-phosphorylation of MCMs with CDKs can stimulate Cdc7 activity, suggesting that CDK phosphorylation may be a prerequisite for Cdc7 function at origins (9).…”

TSPY and its X-encoded homologue interact with cyclin B but exert contrasting functions on cyclin-dependent kinase 1 activities

“…This kit utilized an anti-phospho-Cdc7-Threonine376 monoclonal antibody to detect the CDK1/cyclin B phosphorylation of its specific substrate, Cdc7. Hence, detection of a phosphorylated form of Cdc7 was proportional to the relative amount of activated cyclin B1-CDK1 kinase in the transfected and control cells (Masai et al, 2000). Our results demonstrated that HEK293 cells transfected with the vector alone showed moderate cyclin B1-CDK1 kinase activities.…”

Inhibition of DNA Damage-induced Apoptosis through Cdc7-mediated Stabilization of Tob

“…Kinase Assay-Purified Cdc7-ASK kinase complex (22) was mixed with 2 g of recombinant protein (Tob, proliferating cell nuclear antigen, or MCM2) in kinase buffer (40 mM HepesNaOH, pH 7.4, 10 mM MgCl 2 , 1 mM ␤-glycerophosphate, 1 mM sodium fluoride, 2 mM DTT, 200 M ATP), and reactions were carried out at 30°C for 30 min after the addition of [␥-…”